title: Electrophoretic mobility shift assay of zinc finger proteins: competition for Zn(2+) bound to Sp1 in protocols including EDTA.
abstract: The electrophoretic mobility shift assay (EMSA) offers a principal method to detect specific DNA-protein interactions. As commonly conducted, the reaction and electrophoresis running buffers contain large concentrations of EDTA. EDTA has large affinity for Zn(2+) and readily competes with zinc finger peptides for Zn(2+) resulting in protein unfolding. Nevertheless, EMSA is routinely used to detect zinc finger protein-DNA adducts. This paper examines the chemistry that permits the detection of zinc finger-DNA complexes in the presence of EDTA, using Zn(3)-Sp1 and a cognate DNA binding site, GC1. Twice as much adduct was detected when the reaction was conducted in the absence than in the presence of EDTA. The observation of Zn-Sp1-GC1 was shown to depend on three properties: the inertness of Zn-Sp1-GC1 to reaction with EDTA and the comparatively similar rates of reaction of EDTA and GC1 with Zn(3)-Sp1 under the conditions of the assay that permit some Zn(3)-Sp1-GC1 to form. Inquiring about the mechanism of stabilization of Zn(3)-Sp1 by GC1, EDTA readily reacted with Zn(3)-Sp1 bound to a non-specific DNA, (polydI-dC). Two structurally similar but oppositely charged chelators, nitrilotriacetate (NTA) and tris-(2-ethylaminoethyl) amine (TREN), that react with free Zn(3)-Sp1 failed to compete for zinc bound in the Zn(3)-Sp1-GC-1 adduct. On the basis of these, other results indicated that the stability of Zn(3)-Sp1-GC-1 has a thermodynamic, not a kinetic origin. It is concluded that the observation of zinc finger proteins in the EMSA rests on a fortuitous set of chemical properties that may vary depending on the structures involved.
authors.affiliation: Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53201-0413, USA.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Kothinti
authors.forename: Niloofar M
authors.initials: NM
authors.lastname: Tabatabai
authors.forename: David H
authors.initials: DH
authors.lastname: Petering
mesh_terms: D000818:Animals; D001665:Binding Sites; D004492:Edetic Acid / Q000737:chemistry*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D006801:Humans; D051379:Mice; D016329:Sp1 Transcription Factor / Q000737:chemistry* / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry*; D016335:Zinc Fingers
chemical_list: D016329:Sp1 Transcription Factor; D004492:Edetic Acid; D015032:Zinc